Zhao et al. Virology Journal (2017) 14:114 DOI 10.1186/s12985-017-0781-x RESEARCH Open Access Cyclophilin B facilitates the replication of Orf virus Kui Zhao1, Jida Li2, Wenqi He1, Deguang Song1, Ximu Zhang3, Di Zhang1, Yanlong Zhou1 and Feng Gao1,4* Abstract Background: Viruses interact with host cellular factors to construct a more favourable environment for their efficient replication. Expression of cyclophilin B (CypB), a cellular peptidyl-prolyl cis-trans isomerase (PPIase), was found to be significantly up-regulated. Recently, a number of studies have shown that CypB is important in the replication of several viruses, including Japanese encephalitis virus (JEV), hepatitis C virus (HCV) and human papillomavirus type 16 (HPV 16). However, the function of cellular CypB in ORFV replication has not yet been explored. Methods: Suppression subtractive hybridization (SSH) technique was applied to identify genes differentially expressed in the ORFV-infected MDBK cells at an early phase of infection. Cellular CypB was confirmed to be significantly up- regulated by quantitative reverse transcription-PCR (qRT-PCR) analysis and Western blotting. The role of CypB in ORFV infection was further determined using Cyclosporin A (CsA) and RNA interference (RNAi). Effect of CypB gene silencing on ORFV replication by 50% tissue culture infectious dose (TCID50) assay and qRT-PCR detection. Results: In the present study, CypB was found to be significantly up-regulated in the ORFV-infected MDBK cells at an early phase of infection. Cyclosporin A (CsA) exhibited suppressive effects on ORFV replication through the inhibition of CypB. Silencing of CypB gene inhibited the replication of ORFV in MDBK cells. In conclusion, these data suggest that CypB is critical for the efficient replication of the ORFV genome. Conclusions: Cellular CypB was confirmed to be significantly up-regulated in the ORFV-infected MDBK cells at an early phase of infection, which could effectively facilitate the replication of ORFV. Keywords: ORFV, Cellular cyclophilin B, Cyclosporine A, RNA interference, Replication Background completely unknown. Currently, the host immune re- Orf virus (ORFV) is the type species of the genus Para- sponse to ORFV has been extensively studied, yet many poxvirus, which has a worldwide distribution and is the aspects of the complex host-virus interactions remain causative agent of Orf, a contagious debilitating skin dis- unclear. ease of sheep and goats also known as contagious Cyclophilins (Cyp) comprise a family of peptidyl-prolyl ecthyma, contagious pustular dermatitis, infectious labial cis/trans isomerases, which are originally discovered as a dermatitis, scabby mouth or sore mouth [1]. Primary in- cellular factor with high affinity for the immunosuppres- fections are usually resolved within 1–2 months, how- sant CsA [4, 5]. CypB is one of the most abundant mem- ever repeated and persistent infections can occur in the bers among the Cyp family, is ubiquitously expressed in presence of an intensive inflammatory host antivirus im- most cells, and predominantly resides in the endoplasmic mune response [2, 3]. The mechanisms that establish the reticulum (ER) through the ER retention signal sequence repeated and persistent infections in vivo are almost in the C-terminus [6, 7]. CypB functions in various cellular processes, including transcriptional regulation, protein se- – * Correspondence: [email protected] cretion, immune response and apoptosis [8 10]. In 1College of Veterinary Medicine, Jilin University, 5333 Xi’an Road, Changchun addition, some researchers have shown that many viruses 130062, China such as Japanese encephalitis virus (JEV), hepatitis C virus 4Key Laboratory of Zoonosis, Ministry of Education, College of Veterinary Medicine, Jilin University, 5333 Xi’an Road, Changchun 130062, China (HCV) and human papillomavirus type 16 (HPV 16) Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zhao et al. Virology Journal (2017) 14:114 Page 2 of 11 require CypB for their replication [11–13]. However, there infection, and cell lysates were collected. Normal cell has been no report on the involvement of CypB in the controls were collected in a similar manner. All samples replication of ORFV. were frozen in liquid nitrogen and stored at −80 °C until In the present study, CypB was found to be signifi- SSH library construction. cantly up-regulated in the ORFV-infected MDBK cells at an early phase of infection. However, the function of cel- Construction and screening of Suppression subtractive lular CypB in ORFV replication has not yet been ex- hybridization (SSH) library plored. Sequently, we investigated the role of CypB in Total RNA was isolated from both mock- and ORFV- the replication of ORFV in MDBK cells. Cyclosporin A inoculated cell lysates using RNAiso Plus (Takara, Da- (CsA) exhibited suppressive effects on ORFV replication lian, China) according to the manufacturer’s instructions. through the inhibition of CypB. Silencing of CypB gene The quality of total RNA were determined by ultraviolet inhibited the replication of ORFV in MDBK cells. In spectrophotometry (260 and 280 nm) and 1% agarose/ conclusion, these data suggest that CypB is critical for ethidium bromide (EtBr) gel electrophoresis. Messenger the efficient replication of the ORFV genome. RNA (mRNA) was isolated from total RNA using Poly- ATtract® mRNA Isolation Systems (Promega) according Methods to the manufacturer’s instructions. The purified mRNA Cells and virus was treated with 1 mL of 75% ethanol and 0.1 volume of The Madin―Darby bovine kidney (MDBK) cells were 3 mol/L NaOAc and used as the starting material to maintained in minimal essential medium (MEM) (GIBCO, construct the SSH cDNA library. PCR select cDNA sub- Invitrogen), supplemented with 10% fetal bovine serum traction was conducted using PCR-Select™ cDNA Sub- (FBS) and penicillin (100 U/mL); streptomycin (100 mg/ traction Kit (Clontech, USA) with 2 μg of mRNA for mL); and nystatin (20 mg/mL). The Orf virus used in this each sample as starting material according to the manu- study (ORFV-Jilin) was isolated using MDBK cells from facturer’s instructions. Subtraction hybridization was scab specimens collected from skin lesions of a 6-week- conducted to perform subtraction in both forward and old small-tailed Han sheep afflicted with orf in November reverse directions. After two rounds of hybridization, 2008 in the Jilin province of China [14]. PCR was conducted to selectively amplify cDNAs di- fferentially expressed between ORFV-inoculated and Antibodies and reagents normal MDBK cells. Each PCR amplification described The antibodies used in this study were anti-Cyclophilin B below was conducted using a thermal cycler MyCycler polyconal rabbit antibody (Abcam), PE-Cy5 conjugated (Bio-Rad) in a total volume of 25 μL with 1 μL template rabbit anti-Cyclophilin B antibody (Bioss), anti-β-actin DNA, 0.5 U of 50 × Advantage cDNA polymerase (Proteintech), Peroxidase-conjugated Affinipure Goat (Clontech, USA), 200 μM of each dNTP, and forward Anti-Mouse IgG (Proteintech) and Peroxidase-conjugated and reverse primers (1 μM each). The cDNA fragments Affinipure Goat Anti-Rabbit IgG (Proteintech). CsA were for subtracted secondary PCR products were inserted purchased from Sigma. PolyATtract® mRNA Isolation Sys- into the pMD18-T vector (TaKaRa, Dalian, China) with tems was purchased from Promega. PCR-Select™ cDNA T4 DNA ligase and transformed into DH5a Escherichia Subtraction Kit and Advantage cDNA PCR Kit & Poly- coli cells. The clones with recombinant plasmid were merase Mix were obtained from Clontech. X-treme GENE identified by the LB-ampicillin/IPTG/X-Gal medium HP DNA Transfection Reagent and X-treme GENE siRNA colony screening. Recombinant white clones were se- Transfection Reagent were purchased from Roche. lected randomly and amplified by PCR using the M13 primer to construct the corresponding SSH cDNA li- Virus infection brary. The clones that yielded a single PCR product were MDBK cells have been reported to be least partially per- selected for the next analysis. missive for ORFV replication [14]. A confluent mono- layer of MDBK cells cultured in 75-cm2 flasks were qRT-PCR analysis of CypB expression in ORFV-infected adsorbed with ORFV-Jilin (multiplicity of infection MDBK cells [MOI] = 3) for 2 h at 37 °C. After adsorption, unbound Among the differentially expressed genes obtained by virus was removed by gentle washing with serum-free SSH technique, CypB was found to be significantly up- medium followed by the addition of fresh medium and regulated in the ORFV-infected MDBK cells at an early further incubation at 37 °C. At 2 h after further incuba- phase of infection. The qRT-PCR method based on the tion, ORFV transcripts were detected in MDBK cells in- CypB gene was applied to determine the mRNA expres- fected with ORFV-Jilin. Thus, the starting point of early sion level of CypB in MDBK cells infected with ORFV- phase of ORFV infection was determined for 4 h. The Jilin (MOI = 3) for 0 h, 2 h, 4 h, 8 h, 12 h, 18 h and cultures of incubated cells were harvested at 4 h post 24 h. The primer sets used in the study were specific for Zhao et al. Virology Journal (2017) 14:114 Page 3 of 11 CypB (forward primer 5′-GAGACGGCACTGGAGG- well. Subsequently, the absorbance value at a wavelength of TAAG-3′; reverse primer 5′-TCGTGATGAAGAACT 570 nm was measured using a UV–visible spectrophotom- GGGAG-3′).